This invention relates to methods for making cytokine specific antibodies. This invention also relates to methods for inducing in an animal a high titre antibody response specific for the cytokine. This invention further relates to the treatment of pathological conditions, e.g., eosinophilia, associated with the production of the cytokines. In addition, this invention relates to cytokine conjugates per se and compositions comprising the cytokine conjugates.
Cytokines are involved in many biological functions and are major mediators of the immune response. They are associated with the initiation and progression of various autoimmune diseases. For example, tumor necrosis factor xcex1 (TNFxcex1), interferon xcex3 (IFNxcex3) and interleukin-1 (IL-1) have been associated with diabetes and the destruction of islet cells, the elevated production of Th2 cytokines have been associated with asthma, and interleukin-12 (IL-12) has been associated with rhuematoid arthritis.
Interleukin-9 (xe2x80x9cIL-9xe2x80x9d hereafter), is a glycoprotein which has been isolated from both murine and human cells. See, e.g., U.S. Pat. No. 5,208,218, incorporated by reference. This reference also teaches isolated nucleic acid molecules encoding the protein portion of the molecule, and how to express it.
Since its discovery as a T- and mast cell-growth factor produced by Th2 cells, the physiological processes in which for IL-9 is known to have a role have been gradually expanded (Uyttenhove, et al., Proc. Natl. Acad. Sci. USA 85:6934-6938 (1988); Hxc3xcltner, et al., Eur. J. Immunol. 20:1413-1416 (1990); and Gessner, et al., Immunobiol. 189:419-435 (1993)) (Renauld, J.-C., and Van Snick, J. (1998), The Cytokine Handbook 313-331). Prominent features, disclosed by analysis of transgenic mice overexpressing IL-9, include increased susceptibility to lymphoma-genesis (Renauld, et al., Oncogene 9:1327-1332 (1994)), intestinal mastocytosis (Godfraind, et al., J. Immunol. 160:3989-3996 (1998)), expansion of the B-1 lymphocyte population (Godfraind, et al., J. Immunol. 160:3989-3996 (1998)), bronchial hyper-responsiveness (Temann, et al., J. Exp. Med. 188:1307-1320 (1998), and McLane, et al., Am J. Resp. Cell. Mol. 19:713-720 (1998)) and airway eosinophilia (Dong, et al., Eur. J. Immunol. 29:2130-2139 (1999)). In line with these observations, genetic analyses revealed a linkage between both IL-9 and IL-9 receptor (IL-9R) genes to human asthma (Holroyd, et al., Genomics 52:233-235 (1998) and Marsh, et al. Science 264:1152-1156 (1994)), a finding that was confirmed with respect to IL-9 in murine models (Nicolaides, et al., Proc. Natl. Acad. Sci. USA, 94: 13175-13180 (1997)).
Various uses of IL-9 are disclose in, e.g., U.S. Pat. No.5,164,317 (proliferation of mast cells); U.S. Pat. Nos. 5,246,701 and 5,132,109 (enhancing production of IgG and inhibiting production of IgE), in addition to its first recognized utility, which is as a T cell growth factor. Exemplary of the vast scientific literature on IL-9 are Van Snick, et al, J. Exp. Med. 169(1): 363-368 (1989) (cDNA for the murine molecule, then referred to as P40). Houssiau, et al, J. Immunol 148, (10): 3147-3151 (1992) (IL-2 dependence of IL-9 expression in T lymphocytes). Renauld, et al, Oncogene 9(5):1327-1332 (1994) (effect on thymic lymphomas); Renauld, et al, Blood 85(5):1300-1305 (1995) (anti-apoptotic factor for thymic lymphoma); U.S. Pat. No. 5,830,454 (treatments of cell mediated autoimmune disorders); and U.S. Pat. No.5,935,929 (treating or preventing interstitial lung disease). Review articles may be found at, e.g., Renauld, et al, Cancer Invest, 11(5): 635-640 (1993); Renauld, et al, Adv. Immunol 54:79-97 (1993).
While detrimental in asthma, elevated production of Th2 cytokines has been reported to correlate with resistance to certain parasite infections (Finkelman, et al., Annu. Rev. Immunol., 15:505-533 (1997)). IL-9, for example, was found to enhance mouse resistance to infection with the caecal dwelling nematode T.muris (Faulkner, et al., Infect. Immun., 66:3832-3840 (1998)). This resistance was associated with high IgE and IgG1 levels, as well as with pronounced intestinal mastocytosis.
The absence of T cell help has previously been suggested to be crucial for B cell tolerance toward self-proteins (Dalum, et al., J. Immunol., 157 ; 4786-4804 (1996)). Using bovine luteinizing hormone (LH) as a self protein coupled to ovalbumin (OVA), Johnson, et al. (J. Anim. Sci. 66, 719-726 (1988)) were able to induce high titers of autoantibodies against LH, causing cows to become anestrous. Similarly, a vaccine that prevents pregnancy in women was developed by coupling human chorionic gonadotropin and ovine luteinizing hormone to tetanus and diphtheria toxoids (Talwar, et al., Proc. Natl. Acad. Sci. USA, 91:8532-8536 (1994)). More recently, immunization with a fusion protein of an OVA epitope and mouse TNFxcex1 was found to prevent experimental cachexia and collagen-induced arthritis in mice (Dalum, et al., Nature Biotechnology, 17:666-669 (1999)).
Earlier attempts to generate autoantibodies capable of regulating biological processes, were successfully carried out mainly with hormones (Johnson, et al., J. Anim. Sci., 66:719-726 (1988) and Talwar, et al., Proc. Natl. Acad. Sci. USA, 91:8532-8536 (1994)), hormone receptors (Chackerian, et al. Proc. Natl. Acad. Sci. USA, 96:2773-2778 (1999)) or cellular components (Dong, et al., J. Exp. Med., 179:1243-1252 (1994) and Dalum, et al., Mol. Immunol., 34:1113-1120 (1997)). These observations were recently extended to cytokines with reports of anti-IFNxcex1 induction in AIDS patients (Zagury, et al., Biomed. Pharmacother, 53:90-92 (1999)) and of anti-TNFxcex1 vaccination in mice, the latter preventing cachexia and rheumatoid arthritis (Dalum, et al., Nature Biotechnology, 17:666-669 (1999)).
This invention is directed to methods for making cytokine specific antibodies, particularly antibodies specific for interleukins. This invention is also directed to a method for preventing or treating a pathological condition associated with the production of a cytokine by immunizing an animal with a conjugate of the cytokine and a carrier. Preferably, the cytokine is an interleukin, more preferably, IL-4, IL-5, IL-9, or IL-13 and most preferably the interleukin is IL-9. The IL-9 may be a mammalian IL-9, in particular a murine or human IL-9, preferably a recombinant form or a portion of the IL-9 molecule sufficient to provoke a response.
The invention is also directed to a method for producing prolonged high titre antibodies specific for the cytokine by immunizing a subject with conjugates of the cytokine and a carrier. Preferably, the high titre persists for at least 6 months after the subject is immunized with the conjugate, more preferably, the high titre persists for at least 9 months after the immunization and most preferably, at least 12 months after the immunization.
This invention is further directed to conjugates of a carrier and the cytokine, or portions thereof, and to compositions comprising the conjugates. Preferably, the cytokine is an interleukin and the carrier is ovalbumin (OVA), a substituted OVA, a keyhole limpet hemocyanin (KLH), an acetylated BSA or a pertussis toxin. More preferably the conjugate comprises an interleukin selected from the group consisting of IL-4, IL-5, IL-9 or IL-13, and most preferably comprises IL-9. Especially preferred is a conjugate of IL-9 and ovalbumin. The interleukin may be a recombinant interleukin or an interleukin modified to permit or enhance its conjugation to a carrier.
Those of skill in the art appreciate that many methods are suitable for conjugating a carrier and a conjugation partner. The partners in a conjugate may be treated with a cross-linking agent, e.g., glutaraldehyde, carbodilmide or bis-diazobenzidine. The conjugation partners be also be modified to permit or enhance the formation of conjugates. For example, the carrier may be substituted with maleimide, e.g., a maleimide-substituted OVA, so that it is suitable for forming a conjugate with an interleukin having free SH groups, e.g., a iminothyolane-treated IL-9.
The invention is also directed to methods for producing polyclonal and monoclonal antibodies specific for a cytokine comprising the step of immunizing an animal with a conjugate of the cytokine and a carrier, particularly ovalbumin. In particular the invention is directed to methods for producing monoclonal antibodies specific for IL-9 which comprises immunizing an animal with an IL-9 conjugate, in particular a conjugate of IL-9 and ovalbumin. The monoclonal and polyclonal antibodies of this invention are useful for the treatment of an animal having a disorder that depends at least in part on the production of IL-9.
The invention is also directed to the treatment of a pathological condition or disorder in an animal where the condition or disorder is dependent, at least in part, on the expression of a cytokine, preferably an interleukin, by administering to an animal in need thereof polyclonal or monoclonal antibodies specific to the cytokine. The ability of IL-9 autoantibodies to inhibit the resistance of mice to parasite infection and to abrogate blood eosinophilia is disclosed for the first time herein. Thus a particularly preferred method of this invention is a method for the treatment of eosinophilia and disorders associated with eosinophilia, e.g., allograft rejection, by administering antibodies that are specific for IL-9 to an animal in need thereof. Preferably the animal is a mammal, more preferably the animal is a human.
The methods of this invention also relate to assaying the in vivo effects of long term inhibition of the activity of specific interleukins and for assaying the effectiveness of agents for treating disorders associated with the long term inhibition of an interleukin. For example, one of skill in the art may (1) immunize an animal with an interleukin conjugated with ovalbumin in an amount to induce an immune response that inhibits the activity of the interleukin, (2) assay a parameter associated with a disorder that is dependent on a reduction in the activity of the interleukin before an agent to be assayed is administered to the immunized animal, (3) administer the agent to the immunized animal, (4) assay the parameter after the agent is administered, and (5) compare the parameter before and after the agent is administered to determine the effect of the agent. For example, the level of blood eosinophilia may be assayed in an animal immunized with IL-9xe2x88x92OVA before and after an agent is administered to determine if the agent affects the degree of eosinophilia. Blood eosinophils may be determined by, e.g., counting the cells on slides prepared by centrifugation of 30,000 Ficoll-purified leukocyte cells and staining with, e.g., Diff-Quik (Dade Behring, Deerfield, Ill.).
Those of skill in the art are aware of many methods that are useful for immunizing animals which need not be set forth herein. In the invention described herein, the animals are preferably immunized with 0.5-10 ug of an interleukin conjugate. The interleukin conjugates may be incorporated into any conventional pharmaceutically acceptable vehicle or diluent (see, e.g., Remington""s Pharmaceutical Sciences (19th Ed) (Genarro, ed. (1995) Mack Publishing Co., Easton, Pa.)). For example, the conjugates may be administered with, e.g., buffers, antioxidants such as ascorbic acid, low molecular weight (less than about 10 residues) polypeptides, proteins, amino acids, carbohydrates including glucose, sucrose or dextrins, chelating agents such as EDTA, glutathione and other stabilizers and excipients. Neutral buffered saline or saline mixed with nonspecific serum albumin are exemplary of suitable diluents. The conjugants may also be administered with an adjuvant. Preferred adjuvants are those used routinely in the art, e.g., Freund""s Incomplete Adjuvant or Freund""s Complete Adjuvant and Merck Adjuvant 65. Immunization with the conjugates may also be combined with the administration of other components involved with the recruitment of mast cells, eosinophils or TH2 cells, e.g., MCP-1, MCP-3, MCP-4, Eotaxin, MDC/TARC and I-309, see U.S. Pat. No. 5,824,551.
Preferably, the adjuvantis Freund""s complete or Freund""s incomplete adjuvant and the innoculum comprises about 50% adjuvant (ratio of 1:1, adjuvant:composition). The animal may be immunized with the interleukin conjugate in a plurality of sequential innoculations. Preferably, the innoculations are given every 2 weeks for about 6 weeks. Although any amount of conjugate sufficient to induce an antibody response is suitable for inoculation, preferably each innoculum contains from about lug to about 10 ug conjugate. Preferably, the innoculum contains an amount of about 2 ug of conjugate.